Anal. Chem. 2005, 77, 3660-3670
Measurement of Solution-Phase Chiral Molecular Recognition in the Gas Phase Using Electrospray Ionization-Mass Spectrometry Kevin Schug, Petr Frycˇa´k,† Norbert M. Maier, and Wolfgang Lindner*
Department of Analytical Chemistry and Food Chemistry, University of Vienna, Wa¨hringerstrasse 38, A-1090 Vienna, Austria
Development of chiral selectors (SOs) is important both for understanding chiral molecular recognition processes and for their use in the separation of chiral species (selectands). Their evaluation by chromatographic procedures (e.g., as chiral stationary phase) can, however, be time-consuming. In this respect, electrospray ionization-MS (ESI-MS) is tested here as a possible alternative for screening enantioselective binding by SOs. The set of well-characterized cinchona alkaloid SOs are investigated with respect to their enantioselective binding to a set of model enantiomers, dinitrobenzoyl-(R)- and dinitrobenzoyl-(S)-leucine. MS-based enantioselectivity values from normalized gas-phase ion abundances for the diastereomeric complexes are compared empirically to chromatographic (HPLC) enantioselectivity results and shown to be consistent. Investigations into the fundamentals of measuring unbiased enantioselectivity values in the limit of dilute solution by correlation between experimental and modeled theoretical data are shown. Titration experiments are used to extract binding constants and are compared with published calorimetric (ITC) data. Results show that while the magnitude of binding affinities determined for various diastereomeric complexes is attenuated, the relative ranking and stereochemical preference in binding are consistently reproduced. This work represents a fundamental study of solution- versus gas-phase correlation for enantioselective systems by ESI-MS and indicates that, although not all questions and assumptions can be clearly engaged, for these enthalpically driven binding systems, the relative degree of binding affinity and selectivity is preserved. Chirality, or molecular asymmetry, was first reported by Louis Pasteur in 1848 with the crystallographic analysis of tartaric acid.1 Since this time, and especially in recent years, the phenomenon of chirality has become a central theme in scientific research. The field is driven economically by the pharmaceutical industry (for 9 out of 10 top-selling drugs, accounting for nearly 50 billion dollars gross sales in 2003, the active ingredients are chiral2), but * To whom correspondence should be addressed: E-mail: wolfgang.lindner@ univie.ac.at. Phone: (+43)-1-4277-52300. Fax: (+43)-1-315-1826. † Current address: Department of Analytical Chemistry, Palacky ´ University, trˇ. Svobody 8, 771 46 Olomouc, Czech Republic. (1) Pasteur, L. Comp. Rend. Paris 1848, 26, 535-538. (2) Rouhi, A. M. Chem. Eng. News 2004, 82 (24), 47-62.
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scientifically, it extends further, e.g., to studies about the origin of life3-5 and to the development of optically pure specialty materials.6,7 The common factor between all enantioselective reactions and interactions is the need for a chiral environment. This fundamental concept lies at the heart of the development of chiral selector systems, for example, separation of enantiomeric species and development of chiral catalysts for stereoselective synthesis, important parts of modern chiral molecular recognitionbased research.8 Cinchona alkaloid chiral selector (SO) scaffolds are good examples of molecules that have been developed to effect the separation of enantiomers through chromatographic and electrophoretic processes.9-13 Figure 1 shows the structure of quinine (QN), quinidine (QD), and their O9-carbamate derivatives, which are most commonly employed as SO systems. These selectors can be tethered to solid supports through the alkene double bond to create enantioselective ion exchange-type separation media. Electrostatically driven complexation brings the enantiomerically pure SO and the enantiomers of the selectand (SA) into close contact forming diastereomeric intermediate complexes, where multipoint (or multisite) intermolecular forces, in combination with the chiral environment provided by the SO, allow for one enantiomer of the SA to bind preferentially over the other based on different stability constants. Thus, selectivity for one SA enantiomer relative to the other is a result of the difference in the relative free energy for binding with the selector (∆G(R)-SO‚‚‚(R)-SA - ∆G(R)-SO‚‚‚(S)-SA ) ∆∆G; where “‚‚‚” indicates a noncovalent bond and (R) and (S) indicate pure enantiomers of the respective (3) Schwartz, A. W. Curr. Biol. 1997, 7, R477-R479. (4) Julian, R. R.; Hodyss, R.; Kinnear, B.; Jarrold, M. F.; Beauchamp, J. L. J. Phys. Chem. B 2002, 106, 1219-1228. (5) Takats, Z.; Nanita, S.; Cooks, R. G. Angew. Chem., Int. Ed. 2003, 42, 35213523. (6) Peng, W.; Motonaga, M.; Koe, J. R. J. Am. Chem. Soc. 2004, 126, 1382213826. (7) Xia, H.; Tao, W.; Wang, J.; Zhang, J.; Nie, Q. Opt. Mater. 2004, 27, 279283. (8) Maier, N. M.; Franco, P.; Lindner, W. J. Chromatogr., A 2001, 906, 3-33. (9) Maier, N. M.; Nicoletti, L.; La¨mmerhofer, M.; Lindner, W. Chirality 1999, 11, 522-528. (10) Mandl, A.; Nicoletti, L.; La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 1999, 858, 1-11. (11) Bicker, W.; La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 2004, 1035, 37-46. (12) Lah, J.; Maier, N. M.; Lindner, W.; Vesnaver, G. J. Phys. Chem. B 2001, 105, 1670-1678. (13) Maier, N. M.; Schefzick, S.; Lombardo, G. M.; Feliz, M.; Rissanen, K.; Lindner, W.; Lipkowitz, K. J. Am. Chem. Soc. 2002, 124, 8611-8629. 10.1021/ac050137d CCC: $30.25
© 2005 American Chemical Society Published on Web 04/21/2005
Figure 1. Quinine (8S,9R)- and quinidine (8R,9S)-based cinchona alkaloid chiral selector (SO) derivatives. The base cinchona alkaloid structure is shown here in a generalized salt form.
species, which could, in principle, have one or more chiral centers). In addition, QN- and QD-based SOs exhibit pseudoenantiomeric behavior and, therefore, opposite enantioselectivity for a given pair of SA enantiomers.12,14 To establish the stereoselective molecular recognition mechanism of these SO‚‚‚SA systems, a variety of conventional analytical techniques have been employed. Because these SOs were conceived for application in separations, their use in this context for a variety of SA types, exhibiting large differences in enantioselectivity depending on the structure of both SO and SA, is well-documented.9-11,14-17 Additionally, NMR (NOESY), infrared, circular dichroism, and ultraviolet spectroscopy, as well as microcalorimetric titration, X-ray diffraction, and molecular dynamics calculations, were used to study various aspects of chiral recognition.13,14,18-20 The selector-selectand systems most scrutinized were those incorporating the SOs shown in Figure 1, in combination with the enantiomers of N-3,5-dinitrobenzoylleucine as (R)- or (S)-SAs. NMR job plots showed a clean 1:1 binding stoichiometry.13 Calculations and IR measurements confirmed stabilization of complexes driven by electrostatic binding and enantioselectivity provided by complementary hydrogen bonding, π-π, and steric interactions.19,20 Visual inspection of the alignment of functional units can been made through the analysis of solidphase X-ray diffraction data, shown in Figure 2, for the interaction of DNB-(S)-Leu with chloro-tert-butylcarbamoylquinine.9 In general, analyses of similar SO‚‚‚SA systems under a variety of solution conditions and using a barrage of instrumental techniques show good correlation, enthalpy-driven binding, micromolar dissociation constants, and the capability for obtaining high enantioselectivity values (∆∆G > 6.7 kJ/mol). More than 10 years focused on cinchona alkaloid-derived systems has indeed led to development of diverse, efficient, and useful chiral selector system for acidic analytes, in particular, and (14) La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 1996, 741, 33-48. (15) Chankvetadze, B.; Blaschke, G. J. Chromatogr., A 2001, 906, 309-363. (16) Gasparrini, F.; Misiti, D.; Villani, C. J. Chromatogr., A 2001, 906, 35-50. (17) La¨mmerhofer, M.; Maier, N. M.; Lindner, W. Am. Lab. 1998, 30, 71-78. (18) Czerwenka, C.; Zhang, M. M.; Ka¨hlig, H.; Maier, N. M.; Lipkowitz, K. B.; Lindner, W. J. Org. Chem. 2003, 68, 8315-8327. (19) Wirz, R.; Bu ¨ rgi, T.; Lindner, W.; Baiker, A. Anal. Chem. 2004, 76, 53195330. (20) Lesnik, J.; La¨mmerhofer, M.; Lindner, W. Anal. Chim. Acta 1999, 401, 3-10.
Figure 2. X-ray structure of β-chloro-tert-butylcarbamoyl quinine with DNB-(S)-Leu, depicting the contributions of different binding increments to the formation of enantioselective diastereomeric complexes.9
a wealth of information concerning stereoselective molecular recognition in solution-phase separations, in general.9,10,17,21-25 In retrospect, the evolution of the cinchona alkaloid selector molecules and, for that matter, any selector designed to interact with an analyte of interest in a high affinity and selective fashion could have been achieved much more quickly. The main drawback of evaluating new selector molecules via measurement of chromatographic selectivity factors (R) is the need to synthesize at least 50 mg or greater quantities of an SO compound to be immobilized onto an appropriate support material. The chiral stationary phase (CSP) that is generated must then be packed into a column and evaluated. A significant amount of time can be wasted in the scaleup and evaluation of selector molecules with low affinity or selectivity characteristics in a given selector-selectand system. An alternative approach would be to employ a more sensitive and efficient prescreening technique to pinpoint the most promising selector systems for further evaluation. The most obvious choice is to use soft ionization-mass spectrometry (MS) to attempt to transfer the solution-phase affinity and selectivity measurements for SOs and SAs to gas-phase measurements. If a direct correlation can be established, this approach requires microgram or less quantities of material and a short analysis time to identify promising, and to rule out less promising, candidates for more in-depth inspection. Soft ionization-MS has been used extensively to investigate host-guest (selector-selectand, receptor-ligand, etc.) molecular recognition processes.26-28 Electrospray ionization (ESI) mass (21) Tobler, E.; La¨mmerhofer, M.; Oberleitner, W. R.; Maier, N. M.; Lindner, W. Chromatographia 2000, 51, 65-70. (22) Franco, P.; La¨mmerhofer, M.; Klaus, P. M.; Lindner, W. J. Chromatogr., A 2000, 869, 111-127. (23) Czerwenka, C.; La¨mmerhofer, M.; Maier, N. M.; Rissanen, K.; Lindner, W. Anal. Chem. 2002, 74, 5658-5666. (24) Franco, P.; La¨mmerhofer, M.; Klaus, P. M.; Lindner, W. Chromatographia 2000, 51, 139-146. (25) Krawinkler, K. H.; Maier, N. M.; Ungaro, R.; Sansone, F.; Casnati, A.; Lindner, W. Chirality 2003, 15, S17-S29.
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spectrometry, pioneered by Fenn,29 has enjoyed the most attention in this respect. The types of schemes studied vary greatly, from protein-ligand to macrocyclic encapsulation to chiral discrimination-based systems. These investigations have made use of a variety of solution- and gas-phase-based mass spectrometric methods for obtaining absolute and relative noncovalent binding information.26,27 The ability to obtain information about solution-phase equilibria directly from gas-phase ion abundances is of high interest. This not only applies to the development of accurate screening procedures based on MS but also addresses the use of MS for elucidating important and specific interactions in diverse life science-based settings. In most cases, the molecular recognition process of interest occurs naturally in the solution phase. As such, a discrepancy between interactions in the solution phase (dependent on the specific solvent employed) and those in the gas phase is expected. Molecular recognition systems are commonly referred to in the context of enthalpy/entropy compensation to gauge the contributions of enthalpy (∆H) and entropy (∆S) to the propensity for complex formation or the Gibbs free energy change of the reaction (∆G ) ∆H - T∆S, where T is temperature in kelvin). For a spontaneous reaction to occur, ∆G must be negative. Individual intermolecular forces in the molecular complex contribute in different degrees to ∆H and ∆S and are highly dependent on the nature of the forces and the surrounding medium. Electrostatic forces scale with the inverse of the dielectric of the medium and are enthalpically favored. In particular, hydrogen bonding, an important part of many molecular recognition systems, provides an enthalpic contribution to binding that is highly dependent on the solvation (or lack thereof) present. By their nature, solvophobic forces are also dependent on the type of solvent present but occur, rather, due to entropic stabilization. Upon transferring a complex or a molecular associate from a high dielectric medium, such as water ( ≈ 80), to the gas phase ( ≈ 1), enthalpically favored electrostatic forces become stabilized and entropically favored forces become destabilized. Therefore, the contribution from each force changes to some degree with a phase change, affecting enthalpy/entropy compensation and, presumably, the overall interaction strength that characterizes the binding between the molecules involved. This effect is system dependent and must be considered with each new application. In general, those systems characterized mostly by electrostatic, rather than solvophobic, interactions have the best chance of unbiased survival upon transfer to the gas phase.30 To characterize the solution- to gas-phase correlation, mass spectrometric titration experiments are commonly employed.27,31-35 These are performed by holding the concentration of one (26) Brodbelt, J. S. Int. J. Mass Spectrom. 2000, 200, 57-69. (27) Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Int. J. Mass Spectrom. 2002, 216, 1-27. (28) Schalley, C. Int. J. Mass Spectrom. 2000, 194, 11-39. (29) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4459. (30) Schalley, C. A.; Castellano, R. K.; Brody, M. S.; Rudkevich, D. M.; Siuzdak, G.; Rebek, J., Jr. J. Am. Chem. Soc. 1999, 121, 4568-4579. (31) Sannes-Lowery, K. A.; Griffey, R. H.; Hofstadler, S. A. Anal. Biochem. 2000, 280, 264-271. (32) Loo, J. A.; Hu, P.; McConnell, P.; Mueller, W. T.; Sawyer, T. K.; Thanabal, V. J. Am. Soc. Mass Spectrom. 1997, 8, 234-243. (33) Zhu, M. M.; Rempel, D. L.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2004, 15, 388-397.
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component of a host-guest (selector-selectand) complex constant and varying that of the other component over a wide concentration range (generally, 3 orders of magnitude). The data are then fit to a binding model based on the stoichiometry of association to extract binding constants. This is often achieved through the generation of a Scatchard plot;27,31 however, limitations may exist based on the complexity of the binding process and the experimental observables. Fit of the titration data composed from the gas-phase ion abundances to the binding model employed and correlation to known binding constants measured by alternative solution-phase techniques may provide the best descriptors of qualitative and quantitative compliance for assessing molecular recognition events in the gas phase versus in the solution phase. To test the validity of using MS technology for evaluation of electrostatically stabilized solution-phase chiral molecular recognition systems, a set of well-characterized cinchona alkaloid-based selector-selectand systems are investigated using modern MS methods, employing a quadrupole ion trap mass spectrometer. First, enantioselectivity values and configurational preferences for association between SOs and SAs, screened by MS protocols (assuming measurement in the limit of dilute solution), are compared to those measured in similar aqueous/organic media by HPLC. Second, a 1:1 binding model based on the experimental observables (positive mode ion abundances and initial SO/SA concentrations) is evaluated to study enantioselectivity via the different ion abundances of the diastereomeric complexes. The model is also employed to calculate dissociation constants by curve fitting to data from mass spectrometric titration experiments. These values are compared to those measured by isothermal titration calorimetry (ITC),12 and reasons for discrepancies based on ionization effects are offered. Not only is the potential for expeditious development of the cinchona alkaloid-based enantioselective selector-selectand system by MS realized, but also the application of MS-based titration measurements for extraction of quantitative data and the use of MS in routine screening of molecular recognition is addressed. This work deals with the fundamentals behind a practical application with far-reaching capabilities both for the systems studied here and for gas-phase chiral molecular recognition in general. EXPERIMENTAL SECTION Instrumental. The measurement of gas-phase ions was performed using an Agilent 1100 Series LC/MSD SL ion trap mass spectrometer (Agilent Technologies, Vienna, Austria). Sample mixtures were introduced directly via the LC autosampler and binary pump at 15 µL/min. Positive mode ionization and collection of ions was achieved using the following instrumental parameters: spray capillary voltage (applied to end plate), 5000 V; nebulizer gas pressure, 10 psi; dry gas flow rate, 4 L/min; dry gas temperature, 300 °C; desolvation capillary voltage (end plate offset; unheated transfer line to the high-vacuum region), -500 V; skimmer voltage, 40 V; octapole 1 dc, 12 V; octapole 2 dc varied with SO (from 1.93 to 2.42 V); lens 1, -5 V; and lens 2, -60 V. (34) Zhang, S.; Van Pelt, C. K.; Wilson, D. B. Anal. Chem. 2003, 75, 30103018. (35) Gooding, K. B.; Higgs, R.; Hodge, B.; Stauffer, E.; Heinz, B.; McKnight, K.; Phipps, K.; Shapiro, M.; Winkler, M.; Ng, W.-L.; Julian, R. K. J. Am. Soc. Mass Spectrom. 2004, 15, 884-892.
Partial full scan (100-1500 Th, where 1 Th (Thompson) ) 1 massto-charge (m/z) unit) mass spectra were collected with “enhanced” scan resolution (5500 Th s-1). Each spectrum was generated from an average of 60 scans, where each scan was the average of 5 microscans. Values of ion abundances used for calculation were the average of triplicate measurements. Chemicals. All sample solutions were prepared from standard stock solutions in 50/50 methanol/water (HPLC-grade methanol from Merck (Darmstadt, Germany) and LC/MS-grade ultrapure water from Fluka (Buchs, Switzerland)). The concentration of each component in the final mixtures varied depending on the experiment, but for measurement of the mass spectrometric enantioselectivity values (RMS), SOs and SAs were present at an equimolar concentration of 10 µmol/L (µM). The SOs employed are shown in Figure 1 and include QN (Buchler (Braunschweig, Germany)), QD (Buchler), 2,6-diisopropylphenylcarbamoyl quinine (DIPPCQN; synthesized in-house), 2,6-diisopropylphenylcarbamoyl quinidine (DIPPCQD; synthesized in-house), tert-butylcarbamoylquinine (tBuCQN; synthesized in-house), and tert-butylcarbamoyl quinidine (tBuCQD; synthesized in-house). The SAs investigated were the (R) and (S) enantiomers of N-3,5-dinitrobenzoylleucine (obtained in-house). All materials obtained in-house were used in previous studies and shown to be chemically and enantiomerically pure.10 The background electrolyte strength in each sample was normalized with 10 µM sodium acetate (NaOAc; Fluka) to stabilize sodiated ion abundances. RESULTS AND DISCUSSION Screening Enantioselectivity Using Gas-Phase Ion Abundances. Screening of noncovalent complex formation through soft ionization-mass spectrometric techniques is not a new concept.34-40 Diverse strategies have been applied to diverse systems, including stereoselective binding events; however, little attention has been paid to establishing effective screening procedures for development of chiral selectors. Many methods for measurement of chiral discrimination events by MS exist.27,41-46 Speranza and co-workers have studied the relative stability of diastereomeric ion-molecule aggregates in the gas phase using mass spectrometric and radiolytic techniques.44 Sawada et al. are well known for their stereoselective analysis of crown ether-, carbohydrate-, and antibiotic-based chiral systems using fast atom bombardment.43 Cooks and co-workers have developed the kinetic method and (36) Smith, R. D.; Bruce, J. E.; Wu, Q.; Lei, Q. P. Chem. Soc. Rev. 1997, 26, 191-202. (37) Wigger, M.; Eyler, J. R.; Benner, S. A.; Li, W.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2002, 13, 1162-1169. (38) Min, D.-H.; Tang, W.-J.; Mrksich, M. Nat. Biotechnol. 2004, 22, 717-723. (39) Broeren, M. A. C.; van Dongen, J. L. J.; Pittelkow, M.; Christensen, J. B.; van Genderen, M. H. P.; Meijer, E. W. Angew. Chem., Int. Ed. 2004, 43, 3557-3562. (40) Annis, D. A.; Athanasopoulos, J.; Curran, P. J.; Felsch, J. S.; Kalghatgi, K.; Lee, W. H.; Nash, H. M.; Orminati, J.-P. A.; Rosner, K. E.; Shipps, G. W., Jr.; Thaddupathy, G. R. A.; Tyler, A. N.; Vilenchik, L.; Wagner, C. R.; Wintner, E. A. Int. J. Mass Spectrom. 2004, 238, 77-83. (41) Filippi, A.; Giardini, A.; Piccirillo, S.; Speranza, M. Int. J. Mass Spectrom. 2000, 198, 137-163. (42) Guo, J.; Wu, J.; Siuzdak, G.; Finn, M. G. Angew. Chem., Int. Ed. 1999, 38, 1755-1758. (43) Sawada, M. Mass Spectrom. Rev. 1997, 16, 73-90. (44) Speranza, M. Int. J. Mass Spectrom. 2004, 232, 277-317. (45) Tao, W. A.; Cooks, R. G. Anal. Chem. 2003, 75, 25A-31A. (46) Grigorean, G.; Gronert, S.; Lebrilla, C. B. Int. J. Mass Spectrom. 2002, 219, 79-87.
Figure 3. Enantioselectivity principle based on differential binding between chiral selector (R)-SO and the (R) and (S) enantiomers of the selectand SA. The association reactions measured by mass spectrometry are expressed by the resultant diastereomeric adduct ion abundances.
applied it to determining enantiomeric excess for a variety of amino acids and pharmaceuticals.45 Lebrilla et al. have studied enantioselective guest-exchange reactions between cyclodextrins and amino acids using ion trap mass spectrometry.46 Most of these methods are not, however, amenable to automated setups. A fully automated process necessitates as little operator attendance as possible. Due to the nonchiral environment in a mass spectrometer, methods based on dissociation of diastereomeric complexes that contain a chiral reference molecule (as, for example, in the kinetic method) are most often employed. Other methods, such as those employing elaborate gas-phase ionmolecule or ion-ion reaction procedures require alteration of commercial equipment and also significant tuning. In short, a more simplified approach based on manipulation of ion abundance data collected in partial full-scan mode will be preferred for development of an automated screening protocol, in general, but especially as applied to identification and evaluation of promising chiral selectors. Hypothetically, if the ionization process has no effect on solution-phase equilibria, the degree of complexation of a system can be characterized by the abundance of the ionic noncovalent complex (“adduct ion”) in the gas phase. In practice, this is not the case because of changes in strengths of intermolecular forces upon phase change, equilibrium shifts during ionization, and preferential ionization of some ion forms over others due to different physicochemical attributes.47,48 Still, where chiral discrimination is being measured, a difference in ion abundances for diastereomeric complexes based on two enantiomers (having identical physicochemical properties) interacting with a given chiral selector should be indicative of enantioselective performance by the selector. This statement is qualified only by assuming that the responses of the diastereomeric complexes formed by the two different enantiomers are equal. The validity of this hypothesis cannot be easily verified, although it is an integral assumption for performing these experiments. Measurement of enantioselectivity provided by cinchona-type selectors using MS has been reported previously.49,50 In one investigation, tandem mass spectrometric measurement of enan(47) Cech, N. B.; Enke, C. G. Anal. Chem. 2000, 72, 2717-2723. (48) Sherman, C. L.; Brodbelt, J. S. Anal. Chem. 2003, 75, 1828-1836. (49) Czerwenka, C.; Lindner, W. Rapid Commun. Mass Spectrom. 2004, 18, 2713-2718. (50) Czerwenka, C.; Maier, N. M.; Lindner, W. Anal. Bioanal. Chem. 2004, 379, 1039-1044.
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Figure 4. Mass spectra generated from equimolar mixtures of (A) tBuCQD (SO) and DNB-(R)-Leu (SA) and (B) tBuCQD and DNB-(S)-Leu in 50:50 water/methanol with 10 µM sodium acetate. (For additional experimental conditions, see Experimental Section.) Part B is shown with an inset to illustrate the decreased level of SO‚‚‚SA binding in this system compared to in (A).
tioselective binding between tBuCQN and a series of peptides showed moderate selectivity. In the second, a series of cinchona alkaloid selectors were evaluated for enantioselectivity between DNB-(R)-Ala-(R)-Ala and DNB-(S)-Ala-(S)-Ala from full-scan measurements and compared with liquid-phase separations. Results showed convincing qualitative correlation between HPLC and MS measurements with regard to the relative ranking of the enantioselectivity values. A similar approach is used here to evaluate the enantioselectivity by a series of cinchona alkaloid SOs (Figure 1) for DNB-(R)- and DNB-(S)-Leu. Figure 3 shows the principal of analysis for screening mass spectrometric enantioselectivity (RMS) in this manner. Separate MS experiments using mixtures incorporating 10 µM each of a selector molecule, a pure SA enantiomer, and sodium acetate were performed. This concentration was chosen to provide a reproducible response for the ionic complex of interest and to operate in the range of linear response for the free selector ion (∼0.05-20 µM). Equimolar mixtures prepared at less than 10 µM led to poor reproducibility for some of the weaker binding SO‚‚ ‚SA diastereomeric complexes. Figure 4 shows typical mass spectra and the ion forms of interest. The free SO ion and the sodiated 1:1 ionic SO‚‚‚SA complex ions ([SO + SA + Na]+) were recorded. Neither a free protonated SA molecule nor a protonated 1:1 complex was observed with significant abundance. The RMS values for this experiment were calculated from the ratio of ratios of complexed ([SO + SA + Na]+) to total (free SO + complexed) ion abundances for each SA enantiomer interacting with a given SO. This is shown succinctly by
RMS ) i[(R)-SO+(R)-SA+Na]+/(i[(R)-SO+H]+ + i[(R)-SO+(R)-SA+Na]+) i[(R)-SO+(S)-SA+Na]+/(i[(R)-SO+H]+ + i[(R)-SO+(S)-SA+Na]+)
(1)
where ix indicates the ion abundance of specified ion form (x). 3664 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005
Table 1. Enantioselectivity (N ) 3 for rMS Measurements) and Configurational Preference for Discrimination Measurements Made by HPLC and MS for a Series of Chiral SOs against DNB-(R)-Leu and DNB-(S)-Leua SO
RHPLC
(R)- vs (S)-SA
RMS
(R)- vs (S)-SA
QN QD DIPPCQN DIPPCQD tBuCQN tBuCQD
1.2 1.3 3.5 2.7 15.8 12.5
(R) (S) (S) (R) (S) (R)
1.22 ( 0.06 1.04 ( 0.04 1.56 ( 0.07 2.3 ( 0.1 9.5 ( 0.4 13.1 ( 0.7
(R) nd (S) (R) (S) (R)
a HPLC measurements of R HPLC and configurational preferences were compiled from both the literature12,13,49 and recent (unpublished) measurements (for DIPPCQN and DIPPCQD). HPLC measurements were performed under identical conditions (80:20 methanol/0.1 M ammonium acetate (v/v) adjusted to pH 6). nd, no discrimination observed. Conformational preference ((R)- vs (S)-SA) indicates the enantiomer with the stronger binding affinity for the SO.
Table 1 shows the results of these experiments as well as enantioselectivity values and preferences in enantioselective SA binding measured by HPLC (RHPLC) for the same systems. MS and HPLC measurements are both based on differential binding (diastereomeric complexation) in an aqueous/organic solutionphase system. Still, disparities in experimental conditions, mechanistic aspects, and the methods used for calculating R for each technique permit only empirical correlation between the values and a qualitative ranking of the selector performances. Nevertheless and surprisingly, the magnitude of enantioselectivity values and the binding preferences measured by HPLC and by this MS approach are in good agreement, indicating an empirical correlation between the chiral recognition mechanisms picked up by chromatography (combined adsorption and desorption by enantiomers from a CSP) and that from ESI-MS, involving
the measurement of ionic complexes as a consequence of the transfer from solution to the gas phase. Within each pseudoenantiomeric SO (e.g., QN, DIPPCQN, and tBuCQN), the ranking of enantioselectivity for SA is consistent. Also notable is the reproduction of the reversal of preferred binding configuration with substitution at the O9 position for quinine and its derivatives. These results show that the selected MS method would provide a fairly accurate gauge of the enantioselective performance of cinchona alkaloid selectors when used in an appropriately immobilized form on a support employed in liquid chromatography. Previous works by Cooks and co-workers have also indicated some degree of correlation between measured MS and HPLC enantioselectivities.51,52 Certain discrepancies do, however, exist. First, the RMS values found in this investigation for tBuCQN and tBuCQD are much higher than previously reported for analysis of relatively similar analytes.50 Though such a result is not unwelcome, it points to differences in analytical methodologies that warrant further investigation. This is addressed specifically in the next section and is likely attributed to the sensitivity of the instrument employed here versus in the previously published study. Second, even though configurational preference measured by HPLC and MS is consistent, the degree of enantioselectivity exhibited by pseudoenantiomeric forms of each SO (e.g., tBuCQN and tBuCQD differ only in C8/C9 stereochemistry) varies slightly. This variation is likely tied more intimately to concerted changes in the strengths of intermolecular forces upon transfer of the diastereomeric complexes from the solution to the gas phase. Additionally, only dominant ion abundances were considered for screening RMS values and additional contributions from other ion forms may be significant in some cases. Because the binding between SO and SA in these systems is electrostatically driven and stabilized via the additional intermolecular binding increments contributed by the various binding sites, the intermolecular forces active in the solution-phase formation of diastereomeric complexes are expected to remain intact in the gas phase. This also assumes the ionization process (transfer between the phases) remains “soft” and does not disrupt the binding. Overall, it is desirable to determine and compare the magnitude of individual binding strengths between the selectors and each enantiomer resulting from both solution-phase and MSbased measurement. Moreover, achieving good correlations would provide a sound basis upon which to build MS screening protocols for similar systems. To achieve this, titration measurements based on a 1:1 solution-phase binding model are evaluated. Results of these experiments are presented in a following section. Concentration Dependence in Enantioselective 1:1 Association Processes. Observation of RMS values comparable in magnitude to those obtained through liquid-phase separations was unexpected in light of previous MS investigations of similar systems.50 In the current study, preliminary investigations using sample mixtures with each component present at 50 µM concentration returned more favorable enantioselectivity values than those previously reported (data not shown). Optimization of instrumental variables and sample concentrations was next carried (51) Tao, W. A.; Zhang, D.; Wang, F.; Thomas, P. D.; Cooks, R. G. Anal. Chem. 1999, 71, 4427-4429. (52) Tao, W. A.; Zhang, D.; Nikolaev, E. N.; Cooks, R. G. J. Am. Chem. Soc. 2000, 122, 10598-10609.
out to maximize responses of the ionic complexes. Ideally, measurement of gas-phase ion abundances that correspond to thermodynamic solution-phase binding constants should be performed at the limit of dilute solution conditions. For example, the reported RHPLC values refer to measurements performed in the linear response range in order to avoid overloading of the CSP columns. For MS, the practical limits of operation are governed by saturation of the MS signal at high concentrations and sensitivity at low concentrations. The equimolar sample mixture concentration is minimized with the aim of achieving a reproducible complex ion signal that can be recorded in a linear response region. Lowering the sample mixture concentration revealed a strong dependence of the magnitude of RMS values on sample concentration. In general, dilution of components in the mixture provided greater enantioselectivity, which is in contrast to that observed under linear HPLC conditions. In other words, the MS conditions must also be set so that one approaches the measurement of differential binding affinities in the linear response range of the system. This dilution effect can be reconciled based on the evaluation of a simple 1:1 association model and demonstrates the need for measurement under dilute sample mixture conditions. From eq 2, Ka,(R)
(R)-SO + (R)-SA y\ z (R)-SO‚‚‚(R)-SA K
(2)
d,(R)
an association (Ka,(R)) and dissociation (Kd,(R)) constant can be written as shown in eq 3:
Ka,(R) )
1 Kd,(R)
)
[(R)-SO‚‚‚(R)-SA]eq
(3)
[(R)-SO]eq[(R)-SA]eq
Similar equations can be derived for (S)-SA in order to determine Ka,(S) and Kd,(S) and other related variables of interest, which are defined below for (R)-SA. Based on known quantities (initial concentrations of (R)-SO and (R)-SA enantiomers; denoted as C0,(R)-SO and C0,(R)-SA, respectively) and experimental observables (abundances of free (R)-SO and 1:1 complex ions; i(R)-SO and i(R)-SO‚‚‚(R)-SA, respectively), substitutions to eq 3 can be made to yield an alternate expression for Ka,(R) that fits these quantities:
Ka,(R) ) C0,(R)-SOA(R) (C0,(R)-SO - C0,(R)-SOA(R))(yC0,(R)-SO - C0,(R)-SOA(R))
(4)
Here, A(R) is the degree of association for the (R)-SA enantiomer, given by the equation
A(R) )
[(R)-SO‚‚‚(R)-SA]eq [(R)-SO‚‚‚(R)-SA]eq + [(R)-SO]eq
(5)
and y is the molar ratio of C0,(R)-SA to C0,(R)-SO (y ) 1 for equimolar mixtures; i.e., C0,(R)-SA ) C0,(R)-SO). Equation 4 is quadratic in terms Analytical Chemistry, Vol. 77, No. 11, June 1, 2005
3665
Figure 5. Modeled concentration behavior (dilution curve) of enantioselectivity (R′MS ) A(R)/A(S)) based on a 1:1 binding model and equimolar mixtures. The association constant for (R)-SO‚‚‚(S)-SA (Ka,(S)) is held constant while that for (R)-SO‚‚‚(R)-SA (Ka,(R)) is varied to create an enantioselective system. Model assumes equimolar mixtures (y ) 1) and demonstrates that saturation exists in these binding systems as concentration increases.
of A and can be rearranged to give
Ka,(R)C0,(R)-SOA(R)2 - (Ka,(R)C0,(R)-SOy + Ka,(R)C0,(R)-SO + 1)A(R) + Ka,(R)C0,(R)-SOy ) 0 (6)
Equation 6 can be solved for the degree of association in terms of the association constant and the initial concentration of (R)SO for a particular selector-selectand combination. This provides a means for assessing the concentration dependence of enantioselectivity, assuming only 1:1 complex formation of the diastereomeric associates and no competing pathways. As defined in Figure 3, discrimination of enantiomers occurs when Ka,(R)-SO‚‚‚(R)-SA * Ka,(R)-SO‚‚‚(S)-SA (or congruently, Ka,(R) * Ka,(S)) for the diastereomers. Therefore, the difference in degree of association of enantiomeric analytes ((R)- and (S)-SA) for a given SO in this 1:1 binding model is another measure of enantioselectivity. This can be written succinctly as
R′MS ) A(R)/A(S)
(7)
This is a measure of enantioselectivity different from the RMS value expressed in the previous section and relying directly on the abundance of ions in the MS experiment. Calculation of R′MS here is dependent on the relative percent association by each enantiomer whereas RMS was based solely on the normalized sodiated 1:1 complex ion abundance. By assuming different Ka,(R) and Ka,(S) values, the concentration dependence of R′MS can be modeled graphically. This is shown in Figure 5 for different combinations of Ka,(R) and Ka,(S) (assuming y ) 1), where the (R) enantiomer binds to the selector with the highest affinity. The magnitude and range of values is realistic based on previously measured enantioselectivities and binding constants for this system.12 Assuming Ka,(S) is constant (1 × 104 M-1), Ka,(R) can be varied to describe selector-selectand systems with different levels of enantioselectivity. As the difference in association constants increases, a higher level of enantioselectivity is obtained. Note that only in the limit of very low concentration (dilute solution) 3666 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005
Figure 6. Experimental dilution curve depicting measured enantioselectivity values (between DNB-(R)- and DNB-(S)-Leu) for tBuCQD and DIPPCQD as a function of (equimolar) sample concentration. The higher enantioselectivity exhibited by tBuCQD creates a larger change (3-fold improvement) compared to that with DIPPCQD (2fold improvement) as sample concentration is reduced.
are levels of enantioselectivity that represent true enantioselectivity (i.e., the ratio of association constants) observed. At the upper limit of concentration, R′MS ) 1 and no enantioselectivity is observed. This is due to saturation of binding sites. In practice, for measurement of associative phenomena at a level comparable to those observed in these selector-selectand combinations, and to approach the true levels of enantioselectivity possible, one should work at as low a concentration as possible. Clearly, this level is limited by the sensitivity of the instrument being used. Although the ion trap model is more sensitive than the triple quadrupole system previously employed;50 according to the modeled curves, further improvement could be attained if adduct ions could be detected and reliably integrated using sample concentrations approaching the nanomolar range (
